LOCUS Exported 4298 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pSKDuet01 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4298) AUTHORS Iwai H, Zuger S, Jin J, Tam PH TITLE Highly efficient protein trans-splicing by a naturally split DnaE intein from Nostoc punctiforme. JOURNAL FEBS Lett. 2006 Mar 20. 580(7):1853-8. PUBMED 16516207 REFERENCE 2 (bases 1 to 4298) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4298 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 30..48 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 49..73 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." CDS 129..146 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 156..173 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" promoter 729..747 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 748..772 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." CDS 881..925 /codon_start=1 /product="affinity and epitope tag derived from pancreatic ribonuclease A" /label=S-Tag /translation="KETAAAKFERQHMDS" primer_bind complement(963..981) /label=T7 Term /note="T7 terminator, reverse primer" terminator 977..1024 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" CDS complement(1254..2069) /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind 1977..1996 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" promoter complement(2070..2161) /gene="bla" /label=AmpR promoter rep_origin complement(2177..2926) /direction=LEFT /label=RSF ori /note="Plasmids containing the RSF 1030 origin of replication can be propagated in E. coli cells that contain additional plasmids with compatible origins." primer_bind complement(2187..2204) /label=L4440 /note="L4440 vector, forward primer" primer_bind complement(2427..2446) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3122..4204) /codon_start=1 /gene="lacI" /product="lac repressor" /label=lacI /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" primer_bind 4166..4185 /label=LacI-R /note="LacI, reverse primer" promoter complement(4205..4282) /gene="lacI" /label=lacI promoter ORIGIN 1 tcccttatgc gactcctgca ttaggaaatt aatacgactc actatagggg aattgtgagc 61 ggataacaat tcccctgtag aaataatttt gtttaacttt aataaggaga tataccatgg 121 gcagcagcca tcatcatcat catcacagca gcggcctggt gccgcgcggc agccatatgc 181 agtacaaact tatcctgaac ggtaaaaccc tgaaaggtga aaccaccacc gaagctgttg 241 acgctgctac cgcggaaaaa gttttcaaac agtacgctaa cgacaacggt gttgacggtg 301 aatggaccta cgacgacgct accaaaacct acaccgtaac ggaaggatcc tgtttaagct 361 atgaaacgga aatattgaca gtagaatatg gattattacc gattggtaaa attgtagaaa 421 agcgcatcga atgtactgtt tatagcgttg ataataatgg aaatatttat acacaacctg 481 tagcacaatg gcacgatcgc ggagaacaag aggtgtttga gtattgtttg gaagatggtt 541 cattgattcg ggcaacaaaa gaccataagt ttatgactgt tgatggtcaa atgttgccaa 601 ttgatgaaat atttgaacgt gaattggatt tgatgcgggt tgataatttg ccgaattaag 661 cttgcggccg cataatgctt aagtcgaaca gaaagtaatc gtattgtaca cggccgcata 721 atcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt ccccatctta 781 gtatattagt taagtataag aaggagatat acatatggca gatctcaatt ggatatcggc 841 cggccacgcg atcgctgacg tcggtaccct cgagtctggt aaagaaaccg ctgctgcgaa 901 atttgaacgc cagcacatgg actcgtctac tagcgcagct taattaacct aggctgctgc 961 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt 1021 tttgctgaaa cctcaggcat ttgagaagca cacggtcaca ctgcttccgg tagtcaataa 1081 accggtaaac cagcaataga cataagcggc tatttaacga ccctgccctg aaccgacgac 1141 aagctgacga ccgggtctcc gcaagtggca cttttcgggg aaatgtgcgc ggaaccccta 1201 tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa 1261 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat 1321 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg 1381 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat 1441 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc 1501 ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta 1561 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga 1621 gcgagacgaa atacgcggtc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac 1681 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct 1741 aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga 1801 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg 1861 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct 1921 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg 1981 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag 2041 caagacgttt cccgttgaat atggctcata ctcttccttt ttcaatatta ttgaagcatt 2101 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa 2161 ataggcatgc agcgctcttc cgcttcctcg ctcactgact cgctacgctc ggtcgttcga 2221 ctgcggcgag cggtgtcagc tcactcaaaa gcggtaatac ggttatccac agaatcaggg 2281 gataaagccg gaaagaacat gtgagcaaaa agcaaagcac cggaagaagc caacgccgca 2341 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagcca 2401 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 2461 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 2521 gggaagcgtg gcgctttctc atagctcacg ctgttggtat ctcagttcgg tgtaggtcgt 2581 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 2641 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 2701 cattggtaac tgatttagag gactttgtct tgaagttatg cacctgttaa ggctaaactg 2761 aaagaacaga ttttggtgag tgcggtcctc caacccactt accttggttc aaagagttgg 2821 tagctcagcg aaccttgaga aaaccaccgt tggtagcggt ggtttttctt tatttatgag 2881 atgatgaatc aatcggtcta tcaagtcaac gaacagctat tccgttactc tagatttcag 2941 tgcaatttat ctcttcaaat gtagcacctg aagtcagccc catacgatat aagttgtaat 3001 tctcatgtta gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa 3061 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg 3121 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca 3181 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg 3241 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt 3301 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat 3361 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca 3421 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca 3481 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg 3541 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat 3601 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga 3661 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg 3721 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat 3781 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca 3841 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc 3901 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg 3961 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca 4021 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca 4081 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc 4141 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt 4201 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg 4261 ttttgcgcca ttcgatggtg tccgggatct cgacgctc //